The value of anti-hypertensive drugs in treatment
of primary and secondary hypertension is well established.
Hypertensive disturbances provoke maternal
and fetal morbidity and mortality in 10% of
Toxic effects of drugs may be functional, biochemical,
structural and specific. Such effects are
evaluated by assessment of oxidant–antioxidant
parameters in tissues and severity of DNA damage
The aim of our study is to investigate effects of some antihypertensive drugs (clonidine, methyldopa, amlodipine, ramipril and rilmenidine) on oxidant-antioxidant parameters and DNA in rat uterus tissue. We also histopathologically examine the effects of these antihypertensive drugs on uterus tissue.
Whole biochemical assay compounds were purchased from the following sources: Zdorove Drug, Ukraine (clonidine); Eczacıbaşı Drug, Turkey (methyldopa); Pfizer Drug, Turkey (amlodipine); Aventis Drug, Turkey (ramipril) and Servier Pharmaceuticals, France (rilmenidine).
A total of 36 female healthy adult albino Wistar rats that weighed 205-210 g were obtained from the Ataturk University Medicinal and Experimental Application and Research Center. These rats were divided into six treatment groups before the experimental procedures were initiated: 0.075 mg/kg clonidine group, 100 mg/kg methyldopa group, 2 mg/ kg amlodipine group, 2.5 mg/kg ramipril group, 0.5 mg/kg rilmenidine group and healthy group. Animals were housed and fed under standard conditions in a laboratory where the temperature was kept at 22°C. Animal experiments were performed in accordance with National Guidelines for the Use and Care of Laboratory Animals and approved by the local Animal Care Committee of Ataturk University.
In the experiment, clonidine (0.075 mg/kg), methyldopa
(100 mg/kg), amlodipine (2 mg/kg), ramipril
(2.5 mg/kg) and rilmenidine (0.5 mg/kg) were
administrated to female rats by gastric gavage for
30 days, once a day (
The concentrations of uterus lipid peroxidation
were determined by estimating MDA using the thio
barbituric acid test (
Measurements were performed according to Sun et
al. in uterus tissue (
GPx activity was determined according to the
method of Lawrence and Burk (
The amount of GSH in the total homogenate
was measured according to the method of Sedlak
and Lindsay with some modifications (
DNA isolation from blood was performed according to Miller et al. (
In the hydrolyzed DNA samples, 8-OHdG and dG
levels were measured using high-performance liquid
chromatography (HPLC) with electrochemical
(HPLC-ECD) and variable wavelength detector
(HPLC-UV) systems as described previously (
The specimens were fixed in 10% buffered formalin and routinely processed for paraffin embedding. From each sample, 4 μm thick sections were obtained and stained with hematoxylin eosin to evaluate. The slides were evaluated under light microscopy (Olympus BX51; Olympus Corp., Tokyo, Japan) at 40× magnification. The histopathological evaluation of uterine injury was performed based on parameters that included: degeneration, hemorrhage, edema, vascular proliferation, congestion, neutrophil and eosinophil infiltration. The severity of uterine injury was judged by using a blind semiquantitative scoring system according to previously defined criteria: no injury = 0, mild injury = 1, moderate injury = 2 and severe injury = 3.
All data were subjected to one-way ANOVA using SPSS 13.0 software. Differences between treatment groups and the healthy group were attained using the Scheffe option. Significance was declared at p<0.05 as mean ± SEM.
As in table 1, the following MDA levels were seen: clonidine (0.075 mg/kg), 14.2 ± 0.34; methyldopa (100 mg/kg), 20.8 ± 1.55; amlodipine (2 mg/kg), 25.8 ± 0.94; ramipril (2.5 mg/kg), 41.6 ± 0.98; and rilmenidine (0.5 mg/kg),19.5 ± 0.76 μmol/g. GSH levels were: clonidine (0.075 mg/kg), 9.0 ± 0.4; methyldopa (100 mg/kg), 6.6 ± 0.3; amlodipine (2 mg/kg), 6.1 ± 0.3; ramipril (2.5 mg/kg), 4.2 ± 0.2; and rilmenidine (0.5 mg/kg), 8.1 ± 0.6 U/g. SOD activity was measured as: clonidine (0.075 mg/kg), 6.2 ± 0.35; methyldopa (100 mg/kg), 6.3 ± 0.32; amlodipine (2 mg/kg), 6.8 ± 0.27; ramipril (2.5 mg/kg), 4.5 ± 0.28; and rilmenidine (0.5 mg/ kg), 7.6 ± 0.45 U/g. GPx activity was: clonidine (0.075 mg/kg),11.9 ± 0.73; methyldopa (100 mg/ kg), 7.0 ± 0.47; amlodipine (2 mg/kg), 6.5 ± 0.45; ramipril (2.5 mg/kg), 5.2 ± 0.36; and rilmenidine (05. mg/kg), 8.6 ± 0.86 U/mg. In the healthy rat group, the following values were noted: MDA (11.3 ± 0.26), GSH level (10.6 ± 0.4), SOD (6.4 ± 0.55) and GPx activity (12.5 ± 0.22).
As shown in table 2, the 8-OH Gua/105 Gua levels in the clonidine (0.075 mg/kg), methyldopa (100 mg/kg), amlodipine (2 mg/kg), ramipril (2.5 mg/ kg) and rilmenidine (0.5 mg/kg) groups were: 1.01 ± 0.07, 2.44 ± 0.44, 2.96 ± 0.19, 3.05 ± 0.38 and 2.15 ± 0.10, respectively. This level was 1.21 ± 0.08 in the healthy rat group.
Macroscopically, no alterations occurred in the uteri of the clonidine, methyldopa, amlodipine, ramipril and rilmenidine groups. As seen in table 3 and figures 1-6, microscopic observations of some pathologic parameters such as degeneration, hemorrhage, edema, vascular proliferation, congestion, neutrophil infiltration and eosinophil infiltration showed varying ratios and severities.
Effect of antihypertensive drugs on oxidant and antioxidant parameters in rat uterine tissue. All the treatment groups were compared with the healthy control group
|Drugs||MDA (µmol/g)||p||SOD (U/g)||p||GPx (U/mg)||p||GSH (U/g)||p|
|14.2 ± 0.34||>0.05||6.2 ± 0.35||>0.05||11.9 ± 0.73||>0.05||9.0 ± 0.4||>0.05|
|20.8 ± 1.55||<0.0001||6.3 ± 0.32||>0.05||7.0 ± 0.47||<0.0001||6.6 ± 0.3||<0.0001|
|25.8 ± 0.94||<0.0001||6.8 ± 0.27||>0.05||6.5 ± 0.45||<0.0001||6.1 ± 0.3||<0.0001|
|41.6 ± 0.98||<0.0001||4.5 ± 0.28||<0.05||5.2 ± 0.36||<0.0001||4.2 ± 0.2||<0.0001|
|19.5 ± 0.76||<0.0001||7.6 ± 0.45||>0.05||8.6 ± 0.86||<0.05||8.1 ± 0.6||<0.05|
|11.3 ± 0.26||-||6.4 ± 0.55||-||12.5 ± 0.22||-||10.6 ± 0.4||-|
P values represent differences between the treatment groups and healthy control group. Results are expressed as mean±SEM (n=6).
Effect of antihypertensive drugs on oxidative DNA damage in rat uterine tissue. Treatment groups were compared with healthy control group
|Drugs||Dose (mg/kg)||Animals(n)||DNA damage 8-OH Gua/105 Gua pmol/L||P value|
|0.075||6||1.01 ± 0.07||>0.05|
|100||6||2.44 ± 0.44||<0.05|
|2||6||2.96 ± 0.19||<0.0001|
|2.5||6||3.05 ± 0.38||<0.0001|
|0.5||6||2.15 ± 0.01||<0.05|
|-||6||1.21 ± 0.08||-|
P values represent the significance between the treatment groups and healthy control group. Results are expressed as mean ± SEM
Histopathological examination of clonidine in the rat uterus.
Histopathological examination of amlodipine in the rat uterus.
Histopathological examination of rilmenidine in the rat uterus.
Histopathological examination of methyldopa in the rat uterus.
Histopathological evaluation of uterine injury based on parameters including degeneration, hemorrhage, edema, vascular proliferation, congestion, neutrophil inﬁltration and eosinophils
The severity of uterus injury was judged by using a blind semiquantitative scoring system according to previously deﬁned criteria: no injury = 0, mild injury = +, moderate injury = ++ and severe injury = +++.
Histopathological examination of ramipril in the rat uterus.
Histopathological examination of healthy rat uterus.
In this study, the effects of antihypertensive drug
(clonidine, methyldopa, amlodipine, ramipril
and rilmenidine) treatment on oxidant-antioxidant
parameters and toxic effects on DNA was
investigated in rat uterus tissue. In addition,
uterus tissues were examined histopathologically.
The reason for choosing these doses is that
in a previous study we investigated the effect of
these same doses on ovarian tissue. We determined
that clonidine (0.075 mg/kg) and rilmenidine
(0.5 mg/kg) had no clear negative effect,
methyldopa (100 mg/kg) and amlodipine (2 mg/
kg) had negative effects and ramipril (2.5 mg/
kg) had a severe negative effect on ovarian tissue
of rats (
Oxidants are radicals of oxygen origin that induce
serious damage in tissues (
Ramipril caused a significantly higher repression
of SOD activity than the control group.
There was no significant change in SOD activities
of clonidine, rilmenidine, methyldopa and
amlodipine groups when compared to that of the
control. SOD catalyzes conversion of superoxide
into peroxide and molecular oxygen (
No change in GPX activity of uteri of the clonidine
rats was recorded. Differences between
GPX activities of the clonidine and rilmenidine
groups and the control group were found to be
statistically insignificant. The most meaningful
decrease of GPX activity, when compared to the control group, was observed in the ramipril
group. GPX activities of methyldopa and amlopidin
groups were significantly repressed when
compared to the control group. GPX activity
was found to be lower in damaged tissue than
undamaged tissue, and shown to be proportional
to the severity of damage (
There was no change in GSH levels in the uteri
of the clonidine group. Differences in GSH activity
between clonidine and rilmenidine, and
control groups were statistically insignificant.
The lowest GSH level was observed in the ramipril
group. Previous studies have demonstrated
that GSH levels decrease in damaged tissue
Clonidine is a selective agonist of α2-adrenergic receptors, while rilmenidine is rather a selective agonist of imidazolin l1 receptors. Lack of any negative effects on uterine tissue by clonidine may be attributed to stimulation of α2-adrenergic receptors by clonidine. Amongst other drugs in this study (methyldopa, amlodipine, ramipril), the least toxic on uterine tissue was rilmenidine. This phenomenon may also be due to an agonist effect of rilmenidine on α2-adrenergic receptors. However, the agonist of α2-adrenergic receptors, methyldopa, was found to repress MDA less than clonidine and rilmenidine, but repressed GSH more than clonidine and rilmendin. These negative effects of methyldopa may be attributed to inhibition of dopadecarboxylase enzyme, which converts methyldopa to alpha-methyl-noradrenalin (active metabolite) in noradrenergic neuron terminals. Inhibition of this enzyme hinders formation of the active metabolite (alpha-methylnoradrenalin) of methyldopa.
Oxygen radicals may generate toxic effects on
DNA. Hydroxyl radical causes DNA damage by
removing hydrogen from nucleic acids and reacting
with double bonds (
Under normal circumstances, there is a balance
of oxidative damage and repair in DNA. This
shows that, although very little, there is DNA
damage in healthy individuals, too (
The biochemical toxic effects of these drugs on
uterus tissue were supported via histopathologic
examination which determined the structural
toxic effects of these drugs. Microscopic examinations
showed that histopathologic parameters
such as degeneration, hemorrhage, edema,
vascular proliferation, congestion, neutrophil
infiltration and eosinophil infiltration were seen
the least in the clonidine group and most in the
ramipril group. The degree of these histopathological
data correlated with biochemical toxic
effects. PNL and lymphocyte infiltration show
the severity of drug infiltration (
We divided these drugs into three groups according to their effects on the uterus: (I) mild negative effects (clonidine); (II) moderate negative effects (rilmenidine, methyldopa); and (III) severe negative effects (amlodipine, ramipril). These data may help in the selection of antihypertensive drugs that have the lowest toxicity in pregnant and non-pregnant (pre-pregnancy) women.